Monday, October 27, 2003

Description
Metaphase Block for Cell Synchronization


Procedure
1. Remove the medium from an exponentially-growing cell culture and rinse it with 10 ml of PBS. (See Hint #1)

2. Replace the PBS with Complete Medium with Nocodazole and allow the culture to incubate at 37癈 for 12 hr.

3. Shake off loosely attached, rounded mitotic cells by gently knocking the plate and pipetting medium over the cell layer a few times.

4. Collect the cell-containing medium and place it in a sterile 50 ml polypropylene tube and pellet the cells by centrifugation at 1000 X g for 10 min. Remove the supernatant (the cell pellet may be small) and resuspend the cells in 15 ml of PBS.

5. Repellet the cells at 1000 X g for 10 min and remove the PBS. Resuspend the cells in complete medium and proceed with experiment or return the cells to cell culture flask and incubate at 37癈.


Recipes
PBS pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl


Complete Medium 100 Units/ml Penicillin
100 靏/ml Streptomycin
DMEM
10% (v/v) Calf Serum


Nocodazole Stock (10,000 X) 6 g/ml in DMSO (CAUTION See Hint #2)


Complete Medium with Nocodazole 100 Units/ml Penicillin
Dulbecco's Modified Eagle's Medium (DMEM)
100 靏/ml Streptomycin
10% (v/v) Calf Serum
600 ng/ml Nocodazole (diluted from Nocodazole stock)



Supplies


Tips
1. Different cell lines use different media, thus "complete medium" may be different for your cell line than what is described here.